Compositions comprising bacterial strains

ABSTRACT

The invention provides compositions comprising one or more bacterial strains for treating or preventing visceral hypersensitivity.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/916,202, filed on Mar. 8, 2018 (Pending), which is a continuation ofInternational Application No. PCT/EP2017/025038, filed Mar. 6, 2017,which claims priority to: Great Britain Application No. 1603817.6, filedMar. 4, 2016; Great Britain Application No. 1612191.5, filed Jul. 13,2016; and Great Britain Application No. 1616022.8, filed Sep. 20, 2016;all of which are hereby incorporated by reference in their entirety.Further, all publications, patents, and patent applications mentioned inthis specification are herein incorporated by reference in theirentirety to the same extent as if each individual publication, patent,or patent application was specifically and individually indicated to beincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 4, 2017, isnamed 49455_718_302_Sequence_Listing.txt and is 9,806 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 1500 different phylotypesdominated in abundance levels by two major bacterial divisions (phyla),the Bacteroidetes and the Firmicutes [2-3]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host and additional health benefits. Similarly, the immunologicalimportance of the gut microbiota is well-recognized and is exemplifiedin germfree animals which have an impaired immune system that isfunctionally reconstituted following the introduction of commensalbacteria [4-6].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa and Clostridium clusterXI (F. prausnitzii) bacteria are reduced in IBD patients whilst numbersof E. coli are increased, suggesting a shift in the balance of symbiontsand pathobionts within the gut [7-11].

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[12-15]). A number of strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousbowel disorders (see [16] for a review). Strains of the genus Blautiahave also been proposed for use in modulating the microbial balance ofthe digestive ecosystem (WO 01/85187). However, the relationship betweendifferent bacterial strains and different diseases, and the preciseeffects of particular bacterial strains on the gut and at a systemiclevel and on any particular types of diseases, are poorly characterised.

There is a requirement for the potential effects of gut bacteria to becharacterised so that new therapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating and preventingvisceral hypersensitivity. In particular, the inventors have identifiedthat bacterial strains from the genus Blautia can be effective forreducing visceral hypersensitivity. As described in the examples, oraladministration of compositions comprising Blautia hydrogenotrophica mayreduce visceral hypersensitivity in rat models of visceralhypersensitivity and irritable bowel syndrome (IBS). Therefore, in afirst embodiment, the invention provides a composition comprising abacterial strain of the genus Blautia, for use in a method of treatingor preventing visceral hypersensitivity.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in a methodof treating or preventing visceral hypersensitivity in a subjectdiagnosed with Crohn's disease, ulcerative colitis, functional dyspepsiaor, more preferably, IBS. In other preferred embodiments, the inventionprovides a composition comprising a bacterial strain of the genusBlautia, for use in a method of treating or preventing visceralhypersensitivity in a subject diagnosed with Crohn's disease, ulcerativecolitis, functional dyspepsia, infantile colic or, more preferably, IBS.

In further preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in treatingor preventing visceral hypersensitivity in the abdomen, preferably inthe gastrointestinal tract, and most preferably in the lowergastrointestinal tract. In further embodiments, the compositions of theinvention are for use in treating or preventing visceralhypersensitivity in the caecum, colon or rectum.

In preferred embodiments of the invention, the bacterial strain in thecomposition is of Blautia hydrogenotrophica. Closely related strains mayalso be used, such as bacterial strains that have a 16s rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical tothe 16s rRNA sequence of a bacterial strain of Blautiahydrogenotrophica. Preferably, the bacterial strain has a 16s rRNAsequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to SEQ ID NO:5. Most preferably, the bacterial strain in thecomposition is the Blautia hydrogenotrophica strain deposited underaccession number DSM 10507/14294.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia stercoris. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia stercoris. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 3. Preferably, thesequence identity is to SEQ ID NO:3. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:3.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia wexlerae. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia wexlerae. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or 4. Preferably, thesequence identity is to SEQ ID NO:4. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:4.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for treating visceral hypersensitivity. Also, oraladministration is convenient for patients and practitioners and allowsdelivery to and/or partial or total colonisation of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilised. Lyophilisation is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria, and is shown to provide effectivecompositions in the examples.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of treating or preventingvisceral hypersensitivity, comprising administering a compositioncomprising a bacterial strain of the genus Blautia.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Measurement of BH population by qPCR, showing an increase in BHat days 14 and 28 for animals receiving the BH lyophilisate.

FIG. 2: Impact of BH culture and lypophilisate on animals' response todistension, showing a reduction in contractions for animals receiving BHcompositions.

FIG. 3: Impact of BH culture on microbiota, showing a 1 log decrease inSRB in rats receiving a daily dose of BH culture.

FIG. 4: Impact of BH lyophilisate on microbiota.

FIG. 5: Impact of BH lyophilisate on microbiota fermentation—short chainfatty acids, showing an increase in acetate production in rats treatedwith BH.

FIG. 6: Impact of BH lyophilisate on microbiota fermentation—sulphides,showing a decrease in sulphides production.

FIG. 7: Impact of BH lyophilisate on animals' response to distension.Rats MIH IBS+BH: CRD test.

FIG. 8: Impact of BH lyophilisate on animals' response to distension.Rats MIH IBS+BH: All data—CRD test.

FIGS. 9A-9B: Impact of BH lyophilisate on sulphides. Rats MIH IBS+BH:Sulphides concentrations (mg/L). FIG. 9A illustrates a scatter plot ofthe rat H₂S MIH IBS+BH data. FIG. 9B illustrates a bar graph of the ratH₂S MIH IBS+BH data.

FIGS. 10A-10B: Impact of BH lyophilisate on sulphides. Rats MIH IBS+BH:All data—Sulphides concentrations (mg/L). FIG. 10A illustrates a scatterplot of the rat MIH IBS+BH data.

FIG. 10B illustrates a bar graph of the rat sulphide levels for the MIHIBS+BH data.

FIG. 11: Dosing study in HIM rats—RT-PCR quantification of B.hydrogenotrophica in fecal samples of Healthy HIM rats receivingdifferent concentration of the bacterial species.

FIG. 12: Transit time of B. hydrogenotrophica after oral administration(10⁹/day) to healthy HIM rats.

FIG. 13: Comparison of B. hydrogenotrophica levels found in fecal andcaecal samples of healthy HIM rats (RT-PCR quantification) after 14 daysadministration—B. hydrogenotrophica administrated at 10¹⁰/day/rat.

FIG. 14: Effect of B. hydrogenotrophica (10¹⁰/day for 14 days) on shortchain fatty acids production (RMN ¹H) in caecal contents of healthy HIMrats.

FIG. 15: Impact of B. hydrogenotrophica administration on the microbialpopulations in IBS-HIM rats.

FIG. 16: Sulphides production in IBS-HIM Rats treated with B.hydrogenotrophica (10¹⁰/day for 14 days). Control rats were not treated.

FIG. 17: Changes in patient symptoms during dosing period (days 1-16) ofPhase I clinical trial.

FIG. 18: Changes in patient symptoms during washout period of Phase Iclinical trial.

FIG. 19: qPCR evaluation of B. hydrogenotrophica population in faecalsamples of IBS-HMA rats treated or not with a composition comprising B.hydrogenotrophica (BlautiX) for 28 days.

FIG. 20: Abdominal response to colorectal distension in IBS-HMA ratstreated or not with B. hydrogenotrophica (BlautiX) for 28 days and inuntreated healthy HMA rats.

FIG. 21: Bacteria enumeration in IBS HMA-rat faecal samples after B.hydrogenotrophica (BlautiX) administration versus control solution.

FIG. 22: Sulphide concentration in caecal samples of IBS HMA-ratstreated or not with B. hydrogenotrophica (Blautix) for 28 days.

FIGS. 23A-23B: Short chain fatty acids (SCFA) concentrations in caecalsamples of IBS-HMA rats treated or not with B. hydrogenotrophica(Blautix) for 28 days. FIG. 23A shows concentration of total SCFA. FIG.23B shows concentration of Acetic acid, Propionic acid and Butyric acid.

DISCLOSURE OF THE INVENTION

Bacterial Strains

The compositions of the invention comprise a bacterial strain of thegenus Blautia. The examples demonstrate that bacteria of this genus areuseful for treating or preventing visceral hypersensitivity. Thepreferred bacterial strains are of the species Blautiahydrogenotrophica, Blautia stercoris and Blautia wexlerae. Otherpreferred bacterial strains for use in the invention are Blautiaproducta, Blautia coccoides and Blautia hansenii.

Examples of Blautia strains for use in the invention include Blautiahydrogenotrophica, B. stercoris, B. faecis, B. coccoides, B. glucerasea,B. hansenii, B. luti, B. producta, B. schinkii and B. wexlerae. TheBlautia species are Gram-reaction-positive, non-motile bacteria that maybe either coccoid or oval and all are obligate anaerobes that produceacetic acid as the major end product of glucose fermentation [17].Blautia may be isolated from the human gut, although B. producta wasisolated from a septicaemia sample.

Blautia hydrogenotrophica (previously known as Ruminococcushydrogenotrophicus) has been isolated from the guts of mammals, isstrictly anaerobic, and metabolises H₂/CO₂ to acetate, which may beimportant for human nutrition and health. The type strain of Blautiahydrogenotrophica is S5a33=DSM 10507=JCM 14656. The GenBank accessionnumber for the 16S rRNA gene sequence of Blautia hydrogenotrophicastrain S5a36 is X95624.1 (disclosed herein as SEQ ID NO:5). Thisexemplary Blautia hydrogenotrophica strain is described in [17] and[18]. The S5a33 strain and the S5a36 strain correspond to two subclonesof a strain isolated from a faecal sample of a healthy subject. Theyshow identical morphology, physiology and metabolism and have identical16S rRNA sequences. Thus, in some embodiments, the Blautiahydrogenotrophica for use in the invention has the 16S rRNA sequence ofSEQ ID NO:5.

All deposits were made under the terms of the Budapest Treaty.Maintenance of a viable culture is assured for 30 years from the date ofthe deposit. All restrictions on the availability to the public of thedeposited microorganisms will be irrevocably removed upon the grantingof a patent for this application.

The Blautia hydrogenotrophica bacterium deposited under accession numberDSM 10507 and also under accession number DSM 14294 was tested in theExamples and is also referred to herein as strain BH. Strain BH wasdeposited with the Deutsche Sammlung von Mikroorganismen [GermanMicroorganism Collection] (Mascheroder Weg 1b, 38124 Braunschweig,Germany) in January 1996 as “Ruminococcus hydrogenotrophicus” underaccession number DSM 10507 and also under accession number DSM 14294 as“S5a33” on 10 May 2001. The depositor was INRA Laboratoire deMicrobiologie CR de Clermont-Ferrand/Theix 63122 Saint GenesChampanelle, France. Ownership of the deposits has passed to 4D PharmaPlc by way of assignment.

The GenBank accession number for the 16S rRNA gene sequence of Blautiastercoris strain GAM6-1^(T) is HM626177 (disclosed herein as SEQ IDNO:1). An exemplary Blautia stercoris strain is described in [19]. Thetype strain of Blautia wexlerae is WAL 14507=ATCC BAA-1564=DSM 19850[17]. The GenBank accession number for the 16S rRNA gene sequence ofBlautia wexlerae strain WAL 14507 T is EF036467 (disclosed herein as SEQID NO:2). This exemplary Blautia wexlerae strain is described in [17].

A preferred Blautia stercoris strain is the strain deposited underaccession number NCIMB 42381, which is also referred to herein as strain830. A 16S rRNA sequence for the 830 strain is provided in SEQ ID NO:3.Strain 830 was deposited with the international depositary authorityNCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GTBiologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS,Scotland) on 12 Mar. 2015 as “Blautia stercoris 830” and was assignedaccession number NCIMB 42381. GT Biologics Ltd. subsequently changed itsname to 4D Pharma Research Limited.

A preferred Blautia wexlerae strain is the strain deposited underaccession number NCIMB 42486, which is also referred to herein as strainMRX008. A 16S rRNA sequence for the MRX008 strain is provided in SEQ IDNO:4. Strain MRX008 was deposited with the international depositaryauthority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland)by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen,AB25 2ZS, Scotland) on 16 Nov. 2015 as “Blaqutia/Ruminococcus MRx0008”and was assigned accession number NCIMB 42486.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for treating or preventing visceralhypersensitivity. In certain embodiments, the bacterial strain for usein the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of abacterial strain of Blautia hydrogenotrophica. Preferably, the bacterialstrain for use in the invention has a 16s rRNA sequence that is at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16s rRNA sequence of a bacterial strain ofBlautia stercoris. Preferably, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:1 or SEQ ID NO:3. Preferably,the sequence identity is to SEQ ID NO:3. Preferably, the bacterialstrain for use in the invention has the 16s rRNA sequence represented bySEQ ID NO:3. In certain embodiments, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterialstrain of Blautia wexlerae. Preferably, the bacterial strain for use inthe invention has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or SEQ ID NO:4.Preferably, the sequence identity is to SEQ ID NO:4. Preferably, thebacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:4.

Bacterial strains that are biotypes of the bacterium deposited underaccession number DSM 10507/14294 or biotypes of the bacteria depositedunder accession numbers NCIMB 42381 and NCIMB 42486 are also expected tobe effective for treating or preventing visceral hypersensitivity. Abiotype is a closely related strain that has the same or very similarphysiological and biochemical characteristics.

Strains that are biotypes of a bacterium deposited under accessionnumber DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and that are suitablefor use in the invention may be identified by sequencing othernucleotide sequences for a bacterium deposited under accession numberDSM 10507/14294, NCIMB 42381 or NCIMB 42486. For example, substantiallythe whole genome may be sequenced and a biotype strain for use in theinvention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity across at least 80% of its whole genome (e.g. acrossat least 85%, 90%, 95% or 99%, or across its whole genome). For example,in some embodiments, a biotype strain has at least 98% sequence identityacross at least 98% of its genome or at least 99% sequence identityacross 99% of its genome. Other suitable sequences for use inidentifying biotype strains may include hsp60 or repetitive sequencessuch as BOX, ERIC, (GTG)₅, or REP or [20]. Biotype strains may havesequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity to the corresponding sequence of a bacterium deposited underaccession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486. In someembodiments, a biotype strain has a sequence with at least 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 andcomprises a 16S rRNA sequence that is at least 99% identical (e.g. atleast 99.5% or at least 99.9% identical) to SEQ ID NO:5. In someembodiments, a biotype strain has a sequence with at least 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 andhas the 16S rRNA sequence of SEQ ID NO:5.

Alternatively, strains that are biotypes of a bacterium deposited underaccession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and thatare suitable for use in the invention may be identified by using theaccession number DSM 10507/14294 deposit, the accession number NCIMB42381 deposit, or the accession number NCIMB 42486 deposit, andrestriction fragment analysis and/or PCR analysis, for example by usingfluorescent amplified fragment length polymorphism (FAFLP) andrepetitive DNA element (rep)-PCR fingerprinting, or protein profiling,or partial 16S or 23s rDNA sequencing. In preferred embodiments, suchtechniques may be used to identify other Blautia hydrogenotrophica,Blautia stercoris or Blautia wexlerae strains.

In certain embodiments, strains that are biotypes of a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486 and that are suitable for use in the invention are strains thatprovide the same pattern as a bacterium deposited under accession numberDSM 10507/14294, NCIMB 42381 or NCIMB 42486 when analysed by amplifiedribosomal DNA restriction analysis (ARDRA), for example when usingSau3AI restriction enzyme (for exemplary methods and guidance see, forexample,[21]). Alternatively, biotype strains are identified as strainsthat have the same carbohydrate fermentation patterns as a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486.

Other Blautia strains that are useful in the compositions and methods ofthe invention, such as biotypes of a bacterium deposited under accessionnumber DSM 10507/14294, NCIMB 42381 or NCIMB 42486, may be identifiedusing any appropriate method or strategy, including the assays describedin the examples. For instance, strains for use in the invention may beidentified by culturing bacteria and administering to rats to test inthe distension assay. In particular, bacterial strains that have similargrowth patterns, metabolic type and/or surface antigens to a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486 may be useful in the invention. A useful strain will havecomparable microbiota modulatory activity to the DSM 10507/14294, NCIMB42381 or NCIMB 42486 strain. In particular, a biotype strain will elicitcomparable effects on the visceral hypersensitivity model to the effectsshown in the Examples, which may be identified by using the culturingand administration protocols described in the Examples.

A particularly preferred strain of the invention is the Blautiahydrogenotrophica strain deposited under accession number DSM10507/14294. This is the exemplary BH strain tested in the examples andshown to be effective for treating disease. Therefore, the inventionprovides a cell, such as an isolated cell, of the Blautiahydrogenotrophica strain deposited under accession number DSM10507/14294, or a derivative thereof, for use in therapy, in particularfor the diseases described herein.

A derivative of the strain deposited under accession number DSM10507/14294, NCIMB 42381 or NCIMB 42486 may be a daughter strain(progeny) or a strain cultured (subcloned) from the original. Aderivative of a strain of the invention may be modified, for example atthe genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable microbiota modulatoryactivity to the original DSM 10507/14294, NCIMB 42381 or NCIMB 42486strain. In particular, a derivative strain will elicit comparableeffects on the visceral hypersensitivity model to the effects shown inthe Examples, which may be identified by using the culturing andadministration protocols described in the Examples. A derivative of theDSM 10507/14294 strain will generally be a biotype of the DSM10507/14294 strain. A derivative of the NCIMB 42381 strain willgenerally be a biotype of the NCIMB 42381 strain. A derivative of theNCIMB 42486 strain will generally be a biotype of the NCIMB 42486strain.

References to cells of the Blautia hydrogenotrophica strain depositedunder accession number DSM 10507/14294 encompass any cells that have thesame safety and therapeutic efficacy characteristics as the strainsdeposited under accession number DSM 10507/14294, and such cells areencompassed by the invention. References to cells of the Blautiastercoris strain deposited under accession number NCIMB 42381 encompassany cells that have the same safety and therapeutic efficacycharacteristics as the strains deposited under accession number NCIMB42381, and such cells are encompassed by the invention. References tocells of the Blautia wexlerae strain deposited under accession numberNCIMB 42486 encompass any cells that have the same safety andtherapeutic efficacy characteristics as the strains deposited underaccession number NCIMB 42486, and such cells are encompassed by theinvention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

Therapeutic Uses

In preferred embodiments, the compositions of the invention are for usein treating visceral hypersensitivity. Visceral hypersensitivity is aspecific type of pain characterized by a subjectively painful perceptionlocated in the abdominal area resulting from activation of nociceptorsof the thoracic, pelvic, or abdominal viscera (organs). Visceralhypersensitivity is generally diffuse and difficult to localise, andtherefore contrasts with somatic pain, which is generally sharper andmore localised. Also, visceral hypersensitivity generally is notassociated with specific structural lesions, unlike somatic pain.Visceral nociceptors are intrinsically different from cutaneous and mostother non-visceral nociceptors [22].

Visceral hypersensitivity is generally experienced in the abdomen, butnot all abdominal pain is visceral hypersensitivity. In contrast, thereare many potential causes of abdominal pain and abdominal pain may besomatic, referred or visceral pain. In the abdomen, somatic pain may becaused by an inflamed organ and is generally sharp and localised.Abdominal pain may be caused by fibromyalgia, which is a condition ofsomatic (skin and muscle) hypersensitivity. Referred pain is felt in acutaneous site distant from the diseased organ.

Visceral hypersensitivity is often associated with functional dyspepsiaand irritable bowel syndrome (IBS). However, not all pain associatedwith functional dyspepsia and IBS is visceral hypersensitivity. Indeed,many patients with IBS also exhibit a wide variety of somatic symptomsin abdominal regions (back pain, heartburn) and non-abdominal regions(migraine headaches, dyspareunia, muscle pain in body regionssomatotopically distinct from the gut) [23].

In some embodiments, the pathogenesis of the disease or conditionaffects the intestine. In some embodiments, the pathogenesis of thedisease or condition does not affect the intestine. In some embodiments,the pathogenesis of the disease or condition is not localised at theintestine. In some embodiments, the treating or preventing occurs at asite other than at the intestine. In some embodiments, the treating orpreventing occurs at the intestine and also at a site other than at theintestine. In certain embodiments, the disease or condition is systemic.

Visceral hypersensitivity is also known as visceral pain, and these twoterms are used interchangeably herein.

As demonstrated in the examples, bacterial compositions of the inventionmay be effective for reducing visceral hypersensitivity. In particular,bacterial compositions of the invention can reduce the response tocolorectal distension, which is a manifestation of visceralhypersensitivity that affects many patients. In preferred embodiments,the compositions of the invention are for use in treating or preventingvisceral hypersensitivity in the abdomen, preferably in thegastrointestinal tract, and most preferably in the lowergastrointestinal tract. In further embodiments, the compositions of theinvention are for use in treating or preventing visceralhypersensitivity in the caecum, colon or rectum.

In preferred embodiments, the compositions of the invention are for usein treating or preventing visceral hypersensitivity associated withCrohn's disease, ulcerative colitis, functional dyspepsia, infantilecolic or, more preferably, IBS. In preferred embodiments, thecompositions of the invention are for use in treating or preventingvisceral hypersensitivity in a subject diagnosed with Crohn's disease,ulcerative colitis, functional dyspepsia, infantile colic, or, morepreferably, IBS. In preferred embodiments the compositions of theinvention are for use in treating or preventing visceralhypersensitivity in the treatment of Crohn's disease, ulcerativecolitis, functional dyspepsia, infantile colic, or, more preferably,IBS.

In preferred embodiments, the compositions of the invention are for usein treating or preventing visceral hypersensitivity associated withCrohn's disease, ulcerative colitis, functional dyspepsia or, morepreferably, IBS. In preferred embodiments, the compositions of theinvention are for use in treating or preventing visceralhypersensitivity in a subject diagnosed with Crohn's disease, ulcerativecolitis, functional dyspepsia or, more preferably, IBS. In preferredembodiments the compositions of the invention are for use in treating orpreventing visceral hypersensitivity in the treatment of Crohn'sdisease, ulcerative colitis, functional dyspepsia or, more preferably,IBS. In certain embodiments, the compositions of the invention are foruse in treating visceral hypersensitivity in a patient suffering frompainful distension of the gastrointestinal tract, in particular in thecolon or rectum.

Certain aspects of the discomfort and suffering associated with IBS andother bowel conditions may be caused by the excess production of gasesin the gastrointestinal tract and the bulk volume of these accumulatedgases. The increased volume of different gases may result in flatulence,for example. As shown in the examples, the bacterial compositions of theinvention may be effective for treating a specific aspect of IBS andother bowel conditions—visceral hypersensitivity. Without wishing to bebound by any theory, the observed effect of the bacterial compositionsof the invention on visceral hypersensitivity may be associated with aneffect of the bacteria on a specific gas—H₂S, and an effect on sulphatereducing bacteria (SRB), which synthesise H₂S in the gut. H₂S may haveimportant roles as a pain signalling molecule and the effect of thecompositions of the invention on visceral hypersensitivity observed inthe examples may be related to a reduction in the production of H₂S inthe bowel, which may contribute to visceral hypersensitivity byaffecting pain signalling, independently from any bloating effectsrelated to gas volume. The examples demonstrate that the bacterialcompositions of the invention can be effective for reducing SRB andreducing H₂S. In some embodiments, the bacterial compositions of theinvention reduce SRB and/or reduce H₂S in the caecum. SRB are anaerobicbacteria that use sulphate reduction for the generation of energy andexamples of SRB include members of the genus Desulfovibrio, and inparticular Desulfovibrio piger, which is the most abundant species, andalso the genera Desulfobacter, Desulfobulbus and Desulfotomaculum.

In certain embodiments, the compositions of the invention are for use inreducing colonisation of the gastrointestinal tract by SRB in thetreatment of visceral hypersensitivity. In such embodiments, thecomposition may preferably be in the form of a bacterial culture. Insuch embodiments, the composition may preferably be a lyophilisate. Incertain embodiments, the compositions of the invention are for use inlowering H₂S levels or preventing elevated H₂S levels in thegastrointestinal tract in the treatment of visceral hypersensitivity. Insuch embodiments, the composition may preferably be a lyophilisate.

In certain embodiments, the compositions of the invention are for use inreducing colonisation, community and/or population levels of thegastrointestinal tract by SRB in the treatment of visceralhypersensitivity. In certain embodiments, the compositions of theinvention are for use in reducing colonisation, community and/orpopulation levels of the caecum by SRB in the treatment of visceralhypersensitivity.

In preferred embodiments, the compositions of the invention are for usein reducing colonisation of the gastrointestinal tract by SRB, loweringH₂S levels, or preventing elevated H₂S levels in the treatment ofvisceral hypersensitivity associated with IBS. In further embodiments,the compositions of the invention are for use in reducing colonisationof the gastrointestinal tract by SRB, lowering H₂S levels, or preventingelevated H₂S levels in the treatment of visceral hypersensitivityassociated with Crohn's Disease, ulcerative colitis, functionaldyspepsia or infantile colic, for example in the treatment of visceralhypersensitivity associated with Crohn's Disease, ulcerative colitis orfunctional dyspepsia.

In preferred embodiments, the compositions of the invention are for usein reducing colonisation, community and/or population levels of thegastrointestinal tract by SRB, lowering H2S levels, or preventingelevated H2S levels in the treatment of visceral hypersensitivityassociated with IBS. In further embodiments, the compositions of theinvention are for use in reducing colonisation, community and/orpopulation levels of the gastrointestinal tract by SRB, lowering H2Slevels, or preventing elevated H2S levels in the treatment of visceralhypersensitivity associated with Crohn's Disease, ulcerative colitis,functional dyspepsia or infantile colic, for example, in the treatmentof visceral hypersensitivity associated with Crohn's Disease, ulcerativecolitis or functional dyspepsia.

In preferred embodiments, the compositions of the invention are for usein reducing colonisation of the gastrointestinal tract by SRB, loweringH₂S levels, or preventing elevated H₂S levels in the treatment ofvisceral hypersensitivity in the abdomen, preferably in thegastrointestinal tract, more preferably in the lower gastrointestinaltract, in the caecum, in the colon or in the rectum. In preferredembodiments, the compositions of the invention are for use in reducingcolonisation, community and/or population levels of the gastrointestinaltract by SRB, lowering H2S levels, or preventing elevated H2S levels inthe treatment of visceral hypersensitivity in the abdomen, preferably inthe gastrointestinal tract, more preferably in the lowergastrointestinal tract, in the caecum, in the colon or in the rectum.

In certain embodiments, the compositions of the invention are for use ina method of treating, preventing or reducing colonisation of thegastrointestinal tract by SRB. In certain embodiments, the compositionsof the invention are for use in a method of treating, preventing orreducing colonisation, community and/or population levels of thegastrointestinal tract by SRB. In certain embodiments, the compositionsof the invention are for use in a method of lowering H₂S levels orpreventing elevated H₂S levels in the gastrointestinal tract.

In certain embodiments, the compositions of the invention are for use intreating patients that exhibit, or are expected to exhibit, increasedlevels of SRB and/or H₂S in their gastrointestinal tract, for example,when compared to a healthy subject, or a population of healthy subjects.

In certain embodiments, the compositions of the invention are for use inpreventing visceral hypersensitivity in a subject that is receiving orhas received antibiotic treatment or that is suffering from or hassuffered from bacterial gastroenteritis. Antibiotic treatment andbacterial gastroenteritis are associated with changes in the gutmicrobiota that may precede visceral hypersensitivity and that may beprevented by the compositions of the invention. The compositions of theinvention may be administered concurrently with an antibiotic treatment.

In preferred embodiments, treatment with compositions of the inventionresults in a reduction in visceral hypersensitivity, a reduction incolonisation by SRB, and/or a reduction in H₂S levels.

Treatment or prevention of visceral hypersensitivity may refer to, forexample, an alleviation of the severity of symptoms or a reduction inthe frequency of exacerbations or the range of triggers that are aproblem for the patient. For example, in some embodiments thecomposition of the invention is for use in treating or preventing severevisceral hypersensitivity. In some embodiments the subject having severevisceral hypersensitivity is a subject diagnosed with Crohn's disease,ulcerative colitis, functional dyspepsia, infantile colic, or, morepreferably, IBS. In some embodiments the subject having severe visceralhypersensitivity is a subject diagnosed with Crohn's disease, ulcerativecolitis, functional dyspepsia or, more preferably, IBS.

Modes of Administration

Preferably, the compositions of the invention are to be administered tothe gastrointestinal tract in order to enable delivery to and/or partialor total colonisation of the intestine with the bacterial strain of theinvention. Generally, the compositions of the invention are administeredorally, but they may be administered rectally, intranasally, or viabuccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily. The examples demonstrate that daily administration providessuccessfully colonisation and clinical benefits in the rat model ofvisceral hypersensitivity.

The examples also demonstrate that BH administration may not result inpermanent colonisation of the intestines, so regular administration forextended periods of time may provide greater therapeutic benefits. Thus,the examples show successful delivery of the bacterial strain of theinvention to the colon following daily administration.

Accordingly, in certain embodiments, the compositions of the inventionare administered regularly, such as daily, every two days, or weekly,for an extended period of time, such as for at least one week, twoweeks, one month, two months, six months, or one year.

In some embodiments the compositions of the invention are administeredfor 7 days, 14 days, 16 days, 21 days or 28 days or no more than 7 days,14 days, 16 days, 21 days or 28 days. For example, in some embodimentsthe compositions of the invention are administered for 16 days.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to prevent visceral hypersensitivity developing in her child inutero and/or after it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with visceral hypersensitivity or a disease orcondition associated with visceral hypersensitivity, or that has beenidentified as being at risk of visceral hypersensitivity. Thecompositions may also be administered as a prophylactic measure toprevent the development of visceral hypersensitivity in a healthypatient.

The compositions of the invention may be administered to a patient thathas been identified as having an abnormal gut microbiota. For example,the patient may have reduced or absent colonisation by Blautia, and inparticular Blautia hydrogenotrophica, Blautia stercoris or Blautiawexlerae.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

In some embodiments, the subject to whom the composition is to beadministered is an adult human. In some embodiments, the subject to whomthe composition is to be administered is an infant human.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [24-26]. Theexamples demonstrate that lyophilisate compositions are particularlyeffective. In preferred embodiments, the compositions of the inventioncomprises lyophilised bacteria and is for the treatment of visceralhypersensitivity associated with IBS, preferably for the lowering H₂Slevels or preventing elevated H₂S levels in the treatment of visceralhypersensitivity associated with IBS. In further preferred embodiments,the compositions of the invention comprises lyophilised bacteria and isfor the treatment of visceral hypersensitivity associated with IBS,preferably for use in reducing colonisation of the gastrointestinaltract by SRB in the treatment of visceral hypersensitivity. In furtherpreferred embodiments, the composition of the invention compriseslyophilised bacteria and is for the treatment of visceralhypersensitivity associated with IBS, preferably for use in reducingcolonisation, community and/or population levels of the gastrointestinaltract by SRB in the treatment of visceral hypersensitivity.

Alternatively, the composition of the invention may comprise a live,active bacterial culture. The examples demonstrate that cultures of thebacteria of the invention are therapeutically effective.

In some embodiments, the bacterial strain in the composition of theinvention has not been inactivated, for example, has not beenheat-inactivated. In some embodiments, the bacterial strain in thecomposition of the invention has not been killed, for example, has notbeen heat-killed. In some embodiments, the bacterial strain in thecomposition of the invention has not been attenuated, for example, hasnot been heat-attenuated. For example, in some embodiments, thebacterial strain in the composition of the invention has not beenkilled, inactivated and/or attenuated. For example, in some embodiments,the bacterial strain in the composition of the invention is live. Forexample, in some embodiments, the bacterial strain in the composition ofthe invention is viable. For example, in some embodiments, the bacterialstrain in the composition of the invention is capable of partially ortotally colonising the intestine. For example, in some embodiments, thebacterial strain in the composition of the invention is viable andcapable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of livebacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [27-28].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseBlautia are anaerobes. Other ingredients (such as vitamin C, forexample), may be included as oxygen scavengers and prebiotic substratesto improve the delivery and/or partial or total colonisation andsurvival in vivo. Alternatively, the probiotic composition of theinvention may be administered orally as a food or nutritional product,such as milk or whey based fermented dairy product, or as apharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU; in another example from about 1×10⁷ toabout 1×10¹¹ CFU; in another example from about 1×10⁸ to about 1×10¹⁰CFU; in another example from about 1×10⁸ to about 1×10¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cellsper day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cellsper day.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [29]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [30]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid, cysteine and estersof p-hydroxybenzoic acid, for example, in some embodiments thepreservative is selected from sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused. A further example of a suitable carrier is saccharose. A furtherexample of a preservative is cysteine.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the genus Blautia and do not contain bacteriafrom any other genus, or which comprise only de minimis or biologicallyirrelevant amounts of bacteria from another genus.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism. In some embodiments, such compositionsmay be a lyophilisate that is substantially free from other species oforganism.

In certain embodiments, the compositions of the invention comprise oneor more bacterial strains of the genus Blautia, for example, a Blautiahydrogenotrophica, and do not contain any other bacterial genus, orwhich comprise only de minimis or biologically irrelevant amounts ofbacteria from another genus. In certain embodiments, the compositions ofthe invention comprise a single species of Blautia, for example, aBlautia hydrogenotrophica, and do not contain any other bacterialspecies, or which comprise only de minimis or biologically irrelevantamounts of bacteria from another species. In certain embodiments, thecompositions of the invention comprise a single strain of Blautia, forexample, of Blautia hydrogenotrophica, and do not contain any otherbacterial strains or species, or which comprise only de minimis orbiologically irrelevant amounts of bacteria from another strain orspecies.

In some embodiments, the compositions of the invention comprise morethan one bacterial strain or species. For example, in some embodiments,the compositions of the invention comprise more than one strain fromwithin the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not containbacteria from any other species. In some embodiments, the compositionsof the invention comprise less than 50 strains from within the samespecies (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6,5, 4 or 3 strains), and, optionally, do not contain bacteria from anyother species. In some embodiments, the compositions of the inventioncomprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15,16-25, or 31-50 strains from within the same species and, optionally, donot contain bacteria from any other species. In some embodiments, thecompositions of the invention comprise more than one species from withinthe same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not containbacteria from any other genus. In some embodiments, the compositions ofthe invention comprise less than 50 species from within the same genus(e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or3 species), and, optionally, do not contain bacteria from any othergenus. In some embodiments, the compositions of the invention comprise1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or31-50 species from within the same genus and, optionally, do not containbacteria from any other genus. The invention comprises any combinationof the foregoing.

In some embodiments, the composition comprises a microbial consortium.For example, in some embodiments, the composition comprises the Blautiabacterial strain as part of a microbial consortium. For example, in someembodiments, the Blautia bacterial strain is present in combination withone or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterialstrains from other genera with which it can live symbiotically in vivoin the intestine. For example, in some embodiments, the compositioncomprises a bacterial strain of Blautia hydrogenotrophica in combinationwith a bacterial strain from a different genus. In some embodiments, themicrobial consortium comprises two or more bacterial strains obtainedfrom a faeces sample of a single organism, e.g. a human. In someembodiments, the microbial consortium is not found together in nature.For example, in some embodiments, the microbial consortium comprisesbacterial strains obtained from faeces samples of at least two differentorganisms. In some embodiments, the two different organisms are from thesame species, e.g. two different humans. In some embodiments, the twodifferent organisms are an infant human and an adult human. In someembodiments, the two different organisms are a human and a non-humanmammal.

In some embodiments, the composition of the invention additionallycomprises a bacterial strain that has the same safety and therapeuticefficacy characteristics as the Blautia hydrogenotrophica straindeposited under accession number DSM 10507/14294, but which is not theBlautia hydrogenotrophica strain deposited under accession number DSM10507/14294, or which is not a Blautia hydrogenotrophica or which is nota Blautia.

In some embodiments in which the composition of the invention comprisesmore than one bacterial strain, species or genus, the individualbacterial strains, species or genera may be for separate, simultaneousor sequential administration. For example, the composition may compriseall of the more than one bacterial strain, species or genera, or thebacterial strains, species or genera may be stored separately and beadministered separately, simultaneously or sequentially. In someembodiments, the more than one bacterial strains, species or genera arestored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention isobtained from human adult faeces. In some embodiments in which thecomposition of the invention comprises more than one bacterial strain,all of the bacterial strains are obtained from human adult faeces or ifother bacterial strains are present they are present only in de minimisamounts. The bacteria may have been cultured subsequent to beingobtained from the human adult faeces and being used in a composition ofthe invention.

In some embodiments, the one or more Blautia bacterial strains is/arethe only therapeutically active agent(s) in a composition of theinvention. In some embodiments, the bacterial strain(s) in thecomposition is/are the only therapeutically active agent(s) in acomposition of the invention.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is spray dried. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is live. In certain embodiments, the invention providesthe above pharmaceutical composition, wherein the bacterial strain islyophilised or spray dried and wherein it is viable. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is capable of partially or totally colonising theintestine. In certain embodiments, the invention provides the abovepharmaceutical composition, wherein the bacterial strain is lyophilisedor spray dried and wherein it is viable and capable of partially ortotally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain isreconstituted prior to administration. In some cases, the reconstitutionis by use of a diluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isvisceral hypersensitivity, such as visceral hypersensitivity associatedwith Crohn's disease, ulcerative colitis, functional dyspepsia,infantile colic or, more preferably, IBS.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isvisceral hypersensitivity, such as visceral hypersensitivity associatedwith Crohn's disease, ulcerative colitis, functional dyspepsia or, morepreferably, IBS.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4.0 or about 25.0 and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in asealed container comprising a composition as described herein. In someembodiments, the sealed container is a sachet or bottle. In someembodiments, the composition of the invention is provided in a syringecomprising a composition as described herein.

The composition of the present invention may, in some embodiments, beprovided as a pharmaceutical formulation. For example, the compositionmay be provided as a tablet or capsule. In some embodiments, the capsuleis a gelatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administeredorally. Oral administration may involve swallowing, so that the compoundenters the gastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [31]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [32-34].

The solid or liquid medium used for culture may for example be YCFA agaror YCFA medium. YCFA medium may include (per 100 ml, approximatevalues): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g),cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g),(NH₄)₂SO₄ (0.09 g), MgSO₄.7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin(0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoicacid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg).

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[35-42], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref[43]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref [44].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Efficacy of BacterialInocula in a Rat Model of Visceral Hypersensitivity Summary

Rats were inoculated with the faecal microbiota from a human IBS subjectexhibiting visceral hypersensitivity. The rats were then administeredwith compositions comprising bacterial strains according to theinvention and were then tested using a distension assay to measurevisceral hypersensitivity. The compositions of the invention were foundto reduce the rats' response to distension, indicating a reduction invisceral hypersensitivity.

Strain

Blautia hydrogenotrophica (BH) strain DSM 10507/14294.

Compositions and Administration

BH culture (16H) or lyophilisate—administered by oral gavage

Control solution administered by oral gavage

Rats

Inoculated with human intestinal microbiota from an IBS subject.

Study Design

Day −14—rats inoculated with human intestinal microbiota from an IBSsubject

Days 0 to 28—daily dose of BH culture or lyophilisate, or controlsolution

Days 0, 14 and 28—qPCR of BH population in faecal samples

Between days 14 and 28—operation to implant electrode into the abdomen(for distension assay) Day 28—distension assay, caecal samples collectedfor sulphides and short chain fatty acid (SCFA) analysis, enumeration ofmicrobiota in faecal samples on selective media

Results

FIG. 1 presents the results of a qPCR analysis of the BH population infaecal samples from rats administered control solution (IBS) or BHlyophilisate (IBS+BH). An increase in the BH population was seen at days14 and 28 in rats receiving the BH lyophilisate, which confirmssuccessful colonisation.

FIG. 2 presents the results of the distension assay. Rats were subjectedto colorectal distension and the number of contractions per minute wererecorded as a specific measure of visceral hypersensitivity. The ratstreated with the compositions of the invention exhibited reducedcontractions and reduced visceral hypersensitivity.

FIGS. 3 and 4 report on the effects of administration of BH culture andlyophilisate on the microbiota in faecal samples. Administration of BHculture resulted in a notable reduction (1 log) in sulphate reducingbacteria (SRB).

FIG. 5 reports on the impact of administration of BH lyophilisate onmicrobiota fermentation as measured by short chain fatty acidconcentrations in caecal samples. Administration of BH lyophilisateresulted in an increase in acetate production.

FIG. 6 reports on the impact of administration of BH lyophilisate onmicrobiota fermentation as measured by sulphide concentration in caecalsamples (H₂S). Administration of BH resulted in a decrease in sulphideproduction.

Conclusions

Administration of compositions comprising Blautia hydrogenotrophica ledto successful colonisation and a notable reduction in visceralhypersensitivity, as measured using the distension assay. This effectwas observed when Blautia hydrogenotrophica was administered as aculture and as a lyophilisate. Administration of Blautiahydrogenotrophica also had a notable effect on microbiota constitutionand fermentation, with observed reductions in SRB and sulphideproduction. These data indicate that Blautia hydrogenotrophica may beuseful for reducing visceral hypersensitivity, and in particularvisceral hypersensitivity associated with IBS. The reductions invisceral hypersensitivity may be associated with the observed reductionsin SRB and sulphide production.

Example 2—Efficacy of Bacterial Lyophilisate in a Rat Model of VisceralHypersensitivity

The observations of Example 1 were confirmed in further experimentsusing a lyophilisate of Blautia hydrogenotrophica (BH) strain DSM10507/14294 and a rat model of IBS. As shown in FIGS. 7 and 8,administration of BH lyophilisate provided a statistically-significantreduction in the number of abdominal contractions in response todistension, indicating a reduction in visceral hypersensitivity.Furthermore, as shown in FIGS. 9A, 9B 10A, and 10B, administration of BHlyophilisate provided a statistically-significant reduction insulphides.

Example 3—Effects of Bacterial Lyophilisate on Healthy Rats

The effects of administration of a lyophilisate of Blautiahydrogenotrophica (BH) strain DSM 10507/14294 on healthy HIM rats werestudied and the results are reported in FIGS. 11-14. Further detailsregarding the experiments are provided above in the descriptions of thefigures. FIG. 11 shows that an appropriate dose for BH in rats is 10⁹cells per day or greater. FIG. 12 shows that in these experiments BH didnot permanently colonise the rat digestive tract. FIG. 13 shows that BHis primarily found in the caecum. FIG. 14 shows that administration ofBH induces an increase in acetate as well as in butyrate production.

Example 4—Efficacy of Bacterial Lyophilisate in a Rat Model of VisceralHypersensitivity

The effects of administration of a lyophilisate of Blautiahydrogenotrophica (BH) strain DSM 10507/14294 on a rat model of IBS werefurther investigated. Germ-free rats were inoculated with faecal samplesfrom C-IBS (with constipation) or U-IBS (unsubtyped) patients. Most ofthe experiments were carried out with faecal samples from IBS patientsshowing visceral hypersensitivity (VH measured with barostat). Theresults are reported in FIGS. 15 and 16 and further details regardingthe experiments are provided above in the descriptions of the figures.FIG. 15 confirms that administration of BH lyophilisate causes astatistically-significant reduction in sulphate-reducing bacteria. Asexpected, an increase in BH is also observed. FIG. 16 shows that BHadministration induced a statistically-significant decrease in theamount of H₂S produced by IBS HIM rats. Over-production of caecal H₂S bygut microbiota is associated with visceral hypersensitivity.

Example 5—Changes in Patient Symptoms During Phase I Clinical Trial

A Phase I clinical trial was conducted in which Blautiahydrogenotrophica (“Blautix”, strain deposited under accession numberDSM 10507 and also under accession number DSM 14294) was administered tohuman patients having irritable bowel syndrome (IBS). Patients wereadministered Blautix during a dosing period (days 1-16) with the washoutperiod being day 19-23. Blautix was found to be both safe and welltolerated. Four symptoms were monitored, of which one was abdominalpain. The study recorded whether patients experienced an improvement in,no change in or worsening of each of these symptoms. Results frompatients administered Blautix were compared with those obtained usingpatients administered a placebo. Symptoms were monitored at three timepoints: day 1, day 15/16 and at the end of the study. The results areshown in FIGS. 17 and 18.

When the patients' reported symptoms at day 16 were compared to thebaseline from day 1, 82% of 17 IBS patients receiving Blautix reportedan improvement in symptoms (FIG. 17). Improvement of symptoms, one ofwhich is abdominal pain, supports the use of Blautix for treating orpreventing visceral hypersensitivity. Notably, patients 3.02, 3.17 and3.24, who all had severe abdominal pain at the beginning of the study,had mild, mild, and no abdominal pain, respectively at day 15/16.

50% of patients receiving placebo reported an improvement in symptoms(FIG. 17). High placebo response rates are an established phenomenon inIBS clinical studies. Xifaxan was recently approved to treat IBS basedon much smaller improvements over placebo [45].

A worsening of symptoms at the study completion (day 19-23) compared tosymptoms present upon dosing completion (day 16) is expected based onthe teaching presented here. This worsening of symptoms was seen in thePhase I clinical trial: 41% of IBS patients reported worsening ofsymptoms following cessation of Blautix dosing (FIG. 18). The worseningof symptoms, one of which is abdominal pain, following cessation ofBlautix dosing therefore also supports the use of Blautix in treating orpreventing visceral hypersensitivity.

Example 6—Efficacy of B. Hydrogenotrophica on Visceral HypersensitivityStudied in Human Microbiota Associated Rat (HMA Rat) Model Summary

Groups of 20 germ-free rats were inoculated with the faecal microbiotafrom a human IBS subject (IBS-HMA rats). Three successive experimentswere carried out using faecal samples from 3 different IBS patients. Twoother groups of rats (n=10) were inoculated with faecal samples ofhealthy subject (n=2 subjects; 2 groups of healthy-HMA rats) as visceralsensitivity control. Half of the IBS-HMA rats were then administered for28 days with composition comprising the bacterial strain of B.hydrogenotrophica according to the invention while the other halfanimals received a control solution. After 28 days of administration,all HMA-rats were tested using a colonic distension assay to measurevisceral sensitivity. The composition of the invention were found toreduce the IBS-HMA rats' response to distension, indicating a reductionin visceral hypersensitivity that reached a normo-sensitivity asobserved in healthy-HMA rats.

Strain

Blautia hydrogenotrophica (BH) strain DSM 10507^(T)/14294.

Composition and Administration

BH lyophilisate was suspended in sterile mineral solution to aconcentration of 10¹⁰ bacteria per ml. Two ml of this suspension wasadministered daily per IBS-HMA rat, by oral gavage, for a 28 daysperiod.

The control solution was the sterile mineral solution that wasadministered daily (2 ml per rat) by oral gavage to the control group ofIBS-HMA rats.

Rats

Germ-Free male Fisher rats (aged 10 weeks) were inoculated with humanfaecal microbiota from an IBS subject (IBS-HMA rats). Twenty rats wereinoculated with the same human faecal inoculum. Three successiveexperiments were performed with faecal samples from three different IBSsubjects. Two other groups of ten rats were inoculated with faecalsample from 2 healthy subjects (normo-sensitivity control groups).

Study Design

Day −14—Inoculation of Germ-free rats with human faecal microbiota.

Days 0 to 28—Daily dose of BH lyophilisate (assay group), or controlsolution (control group) by oral gavage

Between days 14 and 22—operation to implant electrode into the abdomen(for distension assay)

Days 22-28—Adaptation of the rats to avoid stress associated withdistension test.

Day 28—distension assay and euthanasia of animals to collect the caecalsamples for sulphides and short chain fatty acid (SCFA) analysis.

Days 0, 14 and 28—Collection of faecal samples for microbial analysis:qPCR for evaluating BH population and other commensal groups ofmiccroorganisms and enumeration of functional groups of microorganismsusing selective media and strictly anaerobic method.

Results

FIG. 19 presents the results of qPCR analysis of the B.hydrogenotrophica population in faecal samples from IBS-HMA ratsreceiving control solution or BH lyophilisate. A significant increase inthe BH population was observed at the end of the administration period(D 28) in rats receiving the BH lyophilisate, which confirms successfuldelivery of BH in the colon.

FIG. 20 presents the results of the distension assay. Rats weresubjected to colorectal distension and the number of contractions per 5minutes were recorded as a specific measure of visceralhypersensitivity. The IBS-HMA rats treated with the composition of theinvention exhibited reduced contractions reflecting a reduction invisceral hypersensitivity. After B. hydrogenotrophica treatment, theIBS-HMA rats showed a normo-visceral sensitivity, comparing with thatmeasured in healthy HMA rats. Three of the rats within the BlautiXtreatment group shown in FIG. 20 are the same as those shown in FIG. 8.

FIG. 21 reports on the effects of administration of B. hydrogenotrophicaon some groups of microorganisms from faecal microbiota, previouslyfound to be affected in IBS patients. Administration of BH resulted in asignificant reduction in sulphate-reducing bacteria (SRB).

FIG. 22 reports on the impact of administration of BH on sulphide (H₂S)concentration in caecal samples of IBS-HMA rats. Administration of BHresulted in a significant decrease in sulphide production. Three of therats within the BlautiX treatment group shown in FIG. 22 are the same asthose shown in FIGS. 10A-10B.

FIG. 23A reports on the impact of administration of BH on the mainfermentative metabolites, short chain fatty acids, in caecal samples ofIBS-HMA rats. Administration of BH-resulted in a significant increase inacetate concentration as well as in a significant increase in butyrateconcentration (FIG. 23B).

Conclusions

Administration of a composition comprising Blautia hydrogenotrophica leda significant reduction in visceral hypersensitivity, as measured usingthe distension assay. After treatment, the visceral sensitivity ofIBS-HMA rats was found to be similar to that measured in healthy-HMArats. Administration of the composition comprising B. hydrogenotrophicacan restore visceral sensitivity of IBS-HMA animals to a normal one.Administration of Blautia hydrogenotrophica also had a significanteffect on microbiota constitution and fermentation, and especiallyinduced important reductions in SRB and sulphide production. These dataindicate that Blautia hydrogenotrophica may be useful for reducingvisceral hypersensitivity, and in particular visceral hypersensitivityassociated with IBS. The reductions in visceral hypersensitivity may beassociated with the observed reductions in SRB and sulphide production.

Example 7—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Sequences(Blautia stercoris strain GAM6-1 16S ribosomal RNA gene, partialsequence - HM626177) SEQ ID NO: 1   1 tgcaagtcga gcgaagcgct tacgacagaa ccttcggggg aagatgtaag ggactgagcg  61 gcggacgggt gagtaacgcg tgggtaacct gcctcataca gggggataac agttggaaac 121 ggctgctaat accgcataag cgcacggtat cgcatgatac agtgtgaaaa actccggtgg 181 tatgagatgg acccgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg 241 atcagtagcc ggcctgagag ggtgaacggc cacattggga ctgagacacg gcccagactc 301 ctacgggagg cagcagtggg gaatattgca caatggggga aaccctgatg cagcgacgcc 361 gcgtgaagga agaagtatct cggtatgtaa acttctatca gcagggaaga aaatgacggt 421 acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca 481 agcgttatcc ggatttactg ggtgtaaagg gagcgtagac ggaagagcaa gtctgatgtg 541 aaaggctggg gcttaacccc aggactgcat tggaaactgt ttttcttgag tgccggagag 601 gtaagcggaa ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa caccagtggc 661 gaaggcggct tactggacgg taactgacgt tgaggctcga aagcgtgggg agcaaacagg 721 attagatacc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg ggagcaaagc 781 tcttcggtgc cgcagcaaac gcaataagta ttccacctgg ggagtacgtt cgcaagaatg 841 aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt taattcgaag 901 caacgcgaag aaccttacca agtcttgaca tcgatctgac cggttcgtaa tggaaccttt 961 ccttcgggac agagaagaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt1021 gggttaagtc ccgcaacgag cgcaacccct atcctcagta gccagcaggt gaagctgggc1081 actctgtgga gactgccagg gataacctgg aggaaggcgg ggacgacgtc aaatcatcat1141 gccccttatg atttgggcta cacacgtgct acaatggcgt aaacaaaggg aagcgagccc1201 gcgaggggga gcaaatccca aaaataacgt cccagttcgg actgcagtct gcaactcgac1261 tgcacgaagc tggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg1321 ggtcttgtac acaccgcccg tcacaccatg ggagtcagta acgcccgaag tc(Blautia wexlerae strain WAL 14507 16S ribosomal RNA gene, partialsequence - EF036467) SEQ ID NO: 2   1 caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg  61 gacgggtgag taacgcgtgg gtaacctgcc ttatacaggg ggataacagt cagaaatggc 121 tgctaatacc gcataagcgc acagagctgc atggctcagt gtgaaaaact ccggtggtat 181 aagatggacc cgcgttggat tagcttgttg gtggggtaac ggcccaccaa ggcgacgatc 241 catagccggc ctgagagggt gaacggccac attgggactg agacacggcc cagactccta 301 cgggaggcag cagtggggaa tattgcacaa tgggggaaac cctgatgcag cgacgccgcg 361 tgaaggaaga agtatctcgg tatgtaaact tctatcagca gggaagatag tgacggtacc 421 tgactaagaa gccccggcta actacgtgcc agcagccgcg gtaatacgta gggggcaagc 481 gttatccgga tttactgggt gtaaagggag cgtagacggt gtggcaagtc tgatgtgaaa 541 ggcatgggct caacctgtgg actgcattgg aaactgtcat acttgagtgc cggaggggta 601 agcggaattc ctagtgtagc ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa 661 ggcggcttac tggacggtaa ctgacgttga ggctcgaaag cgtggggagc aaacaggatt 721 agataccctg gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt ggcaaagcca 781 ttcggtgccg tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg caagaatgaa 841 actcaaagga attgacgggg acccgcacaa gcggtggagc atgtggttta attcgaagca 901 acgcgaagaa ccttaccaag tcttgacatc cgcctgaccg atccttaacc ggatctttcc 961 ttcgggacag gcgagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg1021 gttaagtccc gcaacgagcg caacccctat cctcagtagc cagcatttaa ggtgggcact1081 ctggggagac tgccagggat aacctggagg aaggcgggga tgacgtcaaa tcatcatgcc1141 ccttatgatt tgggctacac acgtgctaca atggcgtaaa caaagggaag cgagattgtg1201 agatggagca aatcccaaaa ataacgtccc agttcggact gtagtctgca acccgactac1261 acgaagctgg aatcgctagt aatcgcggat cagaatgccg cggtgaatac gttcccgggt1321 cttgtacaca ccgcccgtca caccatggga gtcagtaacg cccgaagtca gtgacctaac1381 tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggt(consensus 16S rRNA sequence for Blautia stercoris strain 830)SEQ ID NO: 3TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTACGACAGAACCTTCGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTTGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTGCCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAACCTTTCCTTCGGGACAGAGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAACTGGGGTGAAGTCTAGGGGGT(consensus 16S rRNA sequence for Blautia wexlerae strain MRX008)SEQ ID NO: 4TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTATACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGAGCTGCATGGCTCAGTGTGAAAAACTCCGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCTGTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGGTGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCGCCTGACCGATCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACTGCAAAGAAGGAGCTGCCGAA(Blautia hydrogenotrophica strain S5a36 16S ribosomal RNA gene, partialsequence - X95624.1) SEQ ID NO: 5   1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagcgatag agaacggaga  61 tttcggttga agttttctat tgactgagtg gcggacgggt gagtaacgcg tgggtaacct 121 gccctataca gggggataac agttagaaat gactgctaat accgcataag cgcacagctt 181 cgcatgaagc ggtgtgaaaa actgaggtgg tataggatgg acccgcgttg gattagctag 241 ttggtgaggt aacggcccac caaggcgacg atccatagcc ggcctgagag ggtgaacggc 301 cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg gaatattgca 361 caatggggga aaccctgatg cagcgacgcc gcgtgaagga agaagtatct cggtatgtaa 421 acttctatca gcagggaaga aagtgacggt acctgactaa gaagccccgg ctaattacgt 481 gccagcagcc gcggtaatac gtaaggggca agcgttatcc ggatttactg ggtgtaaagg 541 gagcgtagac ggtttggcaa gtctgatgtg aaaggcatgg gctcaacctg tggactgcat 601 tggaaactgt cagacttgag tgccggagag gcaagcggaa ttcctagtgt agcggtgaaa 661 tgcgtagata ttaggaggaa caccagtggc gaaggcggcc tgctggacgg taactgacgt 721 tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa 781 cgatgaatac taggtgtcgg gtggcaaagc cattcggtgc cgcagcaaac gcaataagta 841 ttcccacctg gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg gggacccgca 901 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aaatcttgac 961 atccctctga ccgggaagta atgttccctt ttcttcggaa cagaggagac aggtggtgca1021 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct1081 tattcttagt agccagcagg tagagctggg cactctaggg agactgccag ggataacctg1141 gaggaaggtg gggatgacgt caaatcatca tgccccttat gatttgggct acacacgtgc1201 tacaatggcg taaacaaagg gaagcgaagg ggtgacctgg agcaaatctc aaaaataacg1261 tctcagttcg gattgtagtc tgcaactcga ctacatgaag ctggaatcgc tagtaatcgc1321 gaatcagaat gtcgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccat1381 gggagtcagt aacgcccgaa gtcagtgacc caaccnaaag gagggagctg ccgaaggtgg1441 gactgataac tggggtga

REFERENCES

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1. A composition comprising a bacterial strain of the genus Blautia, foruse in a method of treating or preventing visceral hypersensitivity. 2.The composition of claim 1, wherein the visceral hypersensitivity isassociated with IBS, Crohn's disease, ulcerative colitis, functionaldyspepsia or infantile colic.
 3. The composition of claim 2, wherein thevisceral hypersensitivity is associated with IBS, Crohn's disease,ulcerative colitis or functional dyspepsia.
 4. The composition of claim1, wherein the composition is for use in treating or preventing visceralhypersensitivity in a subject diagnosed with IBS, Crohn's disease,ulcerative colitis, functional dyspepsia or infantile colic.
 5. Thecomposition of claim 4, wherein the composition is for use in treatingor preventing visceral hypersensitivity in a subject diagnosed with IBS,Crohn's disease, ulcerative colitis or functional dyspepsia.
 6. Thecomposition of claim 1, wherein the composition is for use in reducingcolonisation of the gastrointestinal tract by sulphate reducing bacteria(SRB) in the treatment or prevention of visceral hypersensitivity. 7.The composition of claim 1, wherein the composition is for use inlowering H2S levels or preventing elevated H2S levels in thegastrointestinal tract in the treatment or prevention of visceralhypersensitivity.
 8. The composition of claim 1, wherein the compositionis for use in treating or preventing visceral hypersensitivity in apatient suffering from painful distension of the gastrointestinal tract,in particular in the colon or rectum.
 9. The composition of claim 1,wherein the bacterial strain is of Blautia hydrogenotrophica. 10.(canceled)
 11. (canceled)
 12. The composition of claim 1, wherein thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of abacterial strain of Blautia hydrogenotrophica.
 13. The composition ofclaim 12, wherein the bacterial strain has a 16s rRNA sequence that isat least 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5 or whichhas the 16s rRNA sequence of SEQ ID NO:5.
 14. (canceled)
 15. (canceled)16. The composition of claim 1, wherein the composition comprises abacterial strain of the species Blautia hydrogenotrophica, for use in amethod of treating or preventing visceral hypersensitivity in a subjectdiagnosed with IBS.
 17. The composition of claim 1, wherein thecomposition comprises a bacterial strain of the species Blautiahydrogenotrophica, for use in reducing colonisation of thegastrointestinal tract by sulphate reducing bacteria (SRB), lowering H2Slevels or preventing elevated H2S levels in the gastrointestinal tractin the treatment or prevention of visceral hypersensitivity. 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. Thecomposition of claim 1, wherein the composition is for oraladministration.
 23. The composition of claim 1, wherein the compositioncomprises one or more pharmaceutically acceptable excipients orcarriers.
 24. The composition of claim 1, wherein the bacterial strainis lyophilised.
 25. The composition of claim 1, wherein the bacterialstrain is viable.
 26. The composition of claim 1, wherein thecomposition comprises a single strain of the genus Blautia. 27.(canceled)
 28. (canceled)
 29. (canceled)
 30. A method of treating orpreventing visceral hypersensitivity, comprising administering acomposition comprising a bacterial strain of the genus Blautia to apatient in need thereof.
 31. A method according to claim 30, wherein theBlautia is Blautia hydrogenotrophica.